1、審稿人虐死「實驗小白」。實驗小白初稿中寫到：
第一次修回：Reviewer #2問:Why were the cells fixed after staining? Please provide an explanation (line 12, p7).
實驗小白理直氣壯的回答：The cells were fixed by 1% paraformaldehyde after staining to preserve the cell structure and the specific staining reaction of the mAbs [1, 2]. In addition, fixed cells, previously stained with fluorescein conjugated antibodies, could be stored at 4°C in the dark for at least a week prior to FACS analysis [3]. In our study, the total number of samples (in groups of patients and controls) is about 300, we needed about 24 hours to analyze all these samples by FACSVerse cytometer. Thus, we had to fix the cells with 1% paraformaldehyde to keep the consistency among samples.
1. Berki T, Kumanovics G, Kumanovics A, Falus A, Ujhelyi E, et al. (1998) Production and flow cytometric application of a monoclonal anti-glucocorticoid receptor antibody. J Immunol Methods 214: 19-27.
2. Grutzkau A, Kruger-Krasagakes S, Kogel H, Moller A, Lippert U, et al. (1997) Detection of intracellular interleukin-8 in human mast cells: flow cytometry as a guide for immunoelectron microscopy. J Histochem Cytochem 45: 935-945.
3. Lanier LL, Warner NL (1981) Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. J Immunol Methods 47: 25-30.
第二次修回：很顯然，審稿人對於我們的回答不太滿意，不依不饒的繼續問：Remark about answer to reviewers: the references for fixing after staining are very old and flow cytometry has developed much since. However, fixing might still be valid. Did you check that no tandem dye degradation takes place, by for example looking at CD19 expression in a fully stained sample and in the CD27 FMO? This should give comparable results if the CD27-APC-H7 is not degraded. Also check this for other tandem dyes.
實驗小白無可奈何，只能補做實驗，然後回答審稿人：Indeed, we compared the differences of all tandem dyes with or without fixing within 24h after staining. No significant differences of MFI of tandem dyes were observed in our experiments.
2、審稿人的理由。儘管最後文章還是被接收了，但實驗小白還是被嚇出一身冷汗。到底流式樣本染色後，要不要用多聚甲醛固定？我們查了一些資料（bdbiosciences.com/colors）：
這次審稿人竟然是對的！
至少PE-Cy7和APC-Cy7染色後的樣本在多聚甲醛中只能放置4個小時，而在沒有多聚甲醛的溶液中可存放過夜。
3、染色前固定細胞對流式結果影響巨大我們一般因為以下幾種原因採取先對細胞固定，然後進行流式實驗：便利（很多實驗一天無法做完）、安全（有生物毒性的樣本必須固定殺死細胞後進行實驗）以及胞內染色。然而固定會改變蛋白質的表位，造成結果不準確，甚至錯誤。我們查閱資料發現Biolegend公司做了大量的實驗，比較了不固定和固定（4%多聚甲醛）對流式染色結果的影響。
如下圖所示（黑色表示未染色樣本，紫色表示染色樣本）：
Human (Lysed whole blood/Lymphocytes)Mouse (C57BL/6 Spleen)結果顯示：人外周血來源的淋巴細胞經多聚甲醛固定後，眾多分子的染色水平發生了巨大的變化，尤其是趨化因子受體（如CX3CR1、CXCR3、CCR5和CCR6等）染色出現了非常多的假陽性，從而導致結果不準確，甚至錯誤；與此類似，小鼠脾臟細胞固定後，也出現了眾多分子（如CD40、CD62L、CD4和I-Ab等）染色水平發生顯著變化。(更多內容請查看 http://www.biolegend.com/fixation)
PS:本例中所有數據均來自於Biolegend官方網站，暫未經過實驗小白驗證。
綜上所述，不管是流式染色前還是染色後，為了結果更加準確，最好都不要用多聚甲醛固定樣本。尤其要注意的是，在進行胞內/核內染色的時候，一定要先染表面抗體，然後破膜固定，最後染胞內/核內抗體。切不可為了省事，先破膜固定，然後表面抗體和胞內/核內抗體一起染色。如果您迫不得已一定要固定的話，也一定要先驗證待測分子固定前後染色水平無明顯的變化。
歡迎個人轉發分享，刊物和機構如需轉載，請聯繫授權事宜：shiyanxiaobai@126.com
實驗小白任何穩定性高、重複性好的實驗技術實例都可與我們分享。我們也真誠邀請您撰寫某一領域的前沿和今後的發展方向，為大家的課題設計指明方向！
投稿通道：
1.郵箱:shiyanxiaobai@126.com.
2.添加微信號:Cjer0201.
3.添加qq號:315192331.
PS: 添加qq群號: 227441153（免疫學相關領域），下載相關資源。